Phenoxy thiophene sulfonamides and other compounds for use as inhibitors of bacterial glucuronidase

ABSTRACT

This invention relates generally to compounds that are glucuronidase inhibitors. The glucuronidase inhibitors include phenoxy thiophene sulfonamides, and other compounds such as pyridine sulfonyls, benzene sulfonyls, thiophene sulfonyls, thiazole sulfonyls, thiophene carbonyls, and thiazole carbonyls. These compounds include nialamide, isocarboxazid, phenelzine, amoxapine, loxapine and mefloquine. Also compositions including one or more of such compounds for use in inhibiting glucuronidase and methods of using one or more of such compounds for selective inhibition of bacterial β-glucoronidase. These compounds may be used as a co-drug in combination with the anticancer drug CPT-11. Also a method for screening compounds to determine their usefulness in reducing diarrhea associated with irinotecan chemotherapy.

RELATED APPLICATION INFORMATION

This application is a CONTINUATION of application Ser. No. 15/060,179filed Mar. 3, 2016, which is a CONTINUATION of application Ser. No.13/479,590 filed May 24, 2012 which is a CIP of InternationalApplication PCT/US2011/027974 filed 10 Mar. 2011 entitled “PhenoxyThiophene Sulfonamides And Their Use As Inhibitors Of Glucuronidase”,which was published in the English language on 15 Sep. 2011, withInternational Publication Number WO 2011/112858 A1, and which claimspriority from U.S. Patent Application No. 61/312,512 filed on 10 Mar.2010, the content of which is incorporated herein by reference.

This invention was supported in part by funds from the U.S. Government(National Cancer Institute 04-051311 and National Institutes of Healthgrant 1SC2GM081129). The U.S. Government may have certain rights in theinvention.

This invention relates generally to phenoxy thiophene sulfonamides andother drugs that inhibit bacterial glucuronidase. This invention alsorelates to compositions including one or more of such compounds andmethods of using one or more of such compounds as a co-drug incombination with a camptothecin-derived anticancer drug or other drugthat, in a patient, is metabolized to form a metabolite that is asubstrate for a bacterial β-glucuronidase enzyme. The invention furtherrelates to a method of screening for such compounds. The invention alsoencompasses a method for selectively inhibiting, in a patient to betreated, bacterial-glucuronidase as compared with mammalianβ-glucuronidase, wherein the method comprises administering to thepatient an effective amount of a compound selected from the groupconsisting of nialamide, isocarboxazid, phenelzine, amoxapine, loxapineand mefloquine.

BACKGROUND

Camptothecin, a plant alkaloid derived from the Chinese Camptothecaacuminata tree, was added to the National Cancer Institute's naturalproducts screening set in 1966. It showed strong anti-neoplasticactivity but poor bioavailability and toxic side effects. After thirtyyears of modifying the camptothecin scaffold, two derivatives emergedand are now approved for clinical use. Topotecan (Hycamptin®;GlaxoSmithKline) is currently employed to treat solid ovarian, lung andbrain tumors. CPT-11 (also called Irinotecan, and Camptosar®; Pfizer)contains a carbamate-linked dipiperidino moiety that significantlyincreases bioavailability in mammals. This dipiperidino group is removedfrom the CPT-11 prodrug in vivo by carboxylesterase enzymes thathydrolyze the carbamate linkage to produce the drug's active metabolite,SN-38. CPT-11 is currently used to treat solid colon, lung, and braintumors, along with refractory forms of leukemia and lymphoma.

The sole target of the camptothecins is human topoisomerase I. Thisenzyme relieves superhelical tension throughout the genome and isessential for DNA metabolism, including DNA replication, transcription,and homologous recombination. Topoisomerase I breaks one strand induplex DNA, forming a covalent 3′-phosphotyrosine linkage, and guidesthe relaxation of DNA supercoils. It then reseals the single-strand DNAbreak and releases a relaxed duplex DNA molecule. The camptothecins bindto the covalent topoisomerase I-DNA complex and prevent the religationof the broken single DNA strand, effectively trapping the 91 kDa proteinon the DNA. Such immobilized macromolecular adducts act as roadblocks tothe progression of DNA replication and transcription complexes, causingdouble-strand DNA breaks and apoptosis. Because cancer cells are growingrapidly, the camptothecins impact neoplastic cells more significantlythan normal human tissues. Structural studies have established that thecamptothecins stack into the duplex DNA, replacing the base pairadjacent to the covalent phosphotyrosine linkage. Religation of thenicked DNA strand is prevented by increasing the distance between the5′-hydroxyl and the 3′-phosphotyrosine linkage to >11 Å.

CPT-11 efficacy is severely limited by delayed diarrhea that accompaniestreatment. While an early cholinergic syndrome that generates diarrheawithin hours can be successfully treated with atropine, the diarrheathat appears 2-4 days later is significantly more debilitating anddifficult to control. CPT-11 undergoes a complex cycle of activation andmetabolism that directly contributes to drug-induced diarrhea. CPT-11administered by intravenous injection can traffic throughout the body,but concentrates in the liver where it is activated to SN-38 by thehuman liver carboxylesterase hCE1. The SN-38 generated in the liver isconjugated in the liver to yield SN-38 glucuronide (SN-38G). SN-38G isexcreted from the liver via the bile duct and into the intestines. Oncein the intestines, however. SN-38G serves as a substrate for bacterialglucuronidase enzymes in the intestinal flora that remove theglucuronide moiety and produce the active SN-38. SN-38 in the intestinallumen produced in this manner contributes to epithelial cell death andthe severe diarrhea that limits CPT-11 tolerance and efficacy. Thiseffect has been partially reversed in rats using the relatively weak(IC₅₀=90 μM) β-glucuronidase inhibitor saccharic acid 1,4-lactone.

While broad-spectrum antibiotics have been used to eliminate entericbacteria from the gastrointestinal tract prior to CPT-11 treatment, thisapproach has several drawbacks. First, intestinal flora play essentialroles in carbohydrate metabolism, vitamin production, and the processingof bile acids, sterols and xenobiotics. Thus, the partial or completeremoval of gastrointestinal bacteria is non-ideal for patients alreadychallenged by neoplastic growths and chemotherapy. Second, it is wellestablished that the elimination of the symbiotic gastrointestinal florafrom even healthy patients significantly increases the chances ofinfections by pathogenic bacteria, including enterohemorrhagic E. coliand C. difficile. Third, bacterial antibiotic resistance is a humanhealth crisis, and the unnecessary use of antimicrobials is asignificant contributor to this problem. For these reasons, we pursuedthe targeted inhibition of gastrointestinal bacterial glucuronidasesrather than the broad-spectrum elimination of all enteric microflora.

Glucuronidases hydrolyze glucuronic acid sugar moieties in a variety ofcompounds. The presence of glucuronidases in a range of bacteria isexploited in commonly-used water purity tests, in which the conversionof 4-methylumbelliferyl glucuronide (4-MUG) to 4-methylumbelliferone(4-MU) by glucuronidases is assayed to detect bacterial contamination.Whereas relatively weak inhibitors of glucuronidase have been reported,no potent and/or selective inhibitors of the bacterial enzymes have beenpresented. Thus, there is a need for selective inhibitors of bacterialglucuronidase with a purpose of reducing the dose-limiting side effectand improving the efficacy of the CPT-11 anticancer drug.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to compounds that areeffective as inhibitors of bacterial glucuronidase activity. In thisrespect, the inventors have found that compounds that have GUSinhibitory activity can be used to prevent dose-limiting diarrhea to theirinotecan therapy.

In another aspect, the present invention relates to a compound for usewith camptothecin-derived anticancer drugs. Use of a compound of theinvention with an camptothecin-derived anticancer drug like CPT-11 fortreating cancer reduces the dose-limiting side effects and improves theefficacy of CPT-11. In an aspect of the invention the compound is offormula (I) as described below, which are phenoxy thiophenesulfonamides. In another aspect of the invention, the compound may be apyridine sulfonyl, benzene sulfonyl, thiophene sulfonyl, thiazolesulfonyl, thiophene carbonyl, and/or thiazole carbonyl. In still anotheraspect of the invention, the compound of formula (I), or a compound thatis a pyridine sulfonyl, benzene sulfonyl, thiophene sulfonyl, thiazolesulfonyl, thiophene carbonyl, and/or thiazole carbonyl, is administeredprior to, at the same time as or following administration of CPT-11. Thepresent invention also relates to a method for synthesizing compoundsfor inhibiting glucuronidases. In an aspect of the invention thecompound used of formula (I) as described below, which is a phenoxythiophene sulfonamide. In another aspect of the invention the compoundused may be a pyridine sulfonyl, benzene sulfonyl, thiophene sulfonyl,thiazole sulfonyl, thiophene carbonyl, and/or thiazole carbonyl.

In a further embodiment, the present invention relates to a compound ofthe formula (I):

or a pharmaceutically acceptable salt thereof wherein

each of R₁ and R₂ is the same or different and is selected from H,naphthalene, naphthalene-(C₁-C₄) alkyl, naphthalene-1-ylmethyl,naphthalene-1-ylethyl, naphthalene-1-ylpropyl 3-fluorobenzyl,3-chlorobenzyl 3-bromobenzyl, 3-iodobenzyl, 3-(trifluoromethyl)benzyl,3-(trichloromethyl)benzyl, 3-(tribromomethyl) benzyl,3-(triiodomethyl)benzyl, 3-(C₁-C₄ alkyl)-benzyl, 3-methylbenzyl,3-ethyl-benzyl, 3-propylbenzyl, 3,5-dichlorobenzyl, 3,5-difluorobenzyl,3,5,-dibromobenzyl, 3,5-diiodobenzyl, 3-chlorophenyl, 3-fluorophenyl,3-bromophenyl, 3-iodophenyl, 3-(C₁-C₄ alkyoxy) phenyl, 3-methoxyphenyl,3-ethoxyphenyl, 3-propoxyphenyl, 4-methoxyphenyl, 4-(C₁-C₄ alkyoxy)phenyl, 4-ethoxyphenyl, 4-propoxyphenyl, 2-chlorobenzyl, 3-chlorobenzyl,4-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl,2-bromobenzyl, 3-bromobenzyl, 4-bromobenzyl, 2-iodobenzyl, 3-iodobenzyl,4-iodobenzyl, 3-(C₁-C₄ alkyoxy) benzyl; 3-methoxybenzyl,4-methoxy-benzyl, 3-ethoxybenzyl, 4-ethoxybenzyl, 3-propoxybenzyl,4-(C₁-C₄ alkyoxy)phenyl and 4 propoxybenzyl,

each of R₃ and R₄ is the same or different and is selected from H, F,Cl, Br, and I, and

R₅ is selected from 3-(R-1-yl)phenyl, and 4-(R-1-yl)phenyl, wherein R isselected from piperazin, 4-(C₁-C₄ alkyl) piperazin, 4-methylpiperazin,4-ethyl-piperazin, and 4-propylpiperazin.

In yet another embodiment, the invention relates to a method forinhibiting or reducing diarrhea in a patient being treated with a drugthat gets metabolized to form a metabolite that is a substrate for abacterial β-glucuronidase enzyme. The method comprises administering tothe patient a compound in an amount effective to inhibit the bacterialβ-glucuronidase enzyme, wherein the compound is selected from the groupconsisting of nialamide, isocarboxazid, phenelzine, amoxapine, andmefloquine. In a preferred embodiment, the drug is irinotecan.

In another aspect, the present invention also relates to a method forinhibiting bacterial β-glucuronidase in a subject in need thereof whichcomprises administering to the subject one or more compounds thatinhibit the glucuronidase. In an aspect of the invention the compound isof formula (I) as described below, which is a phenoxy thiophenesulfonamide. In another aspect of the invention the compound may be apyridine sulfonyl, benzene sulfonyl, thiophene sulfonyl, thiazolesulfonyl, thiophene carbonyl, and/or thiazole carbonyl.

In another aspect of the invention, the compound is one whichselectively inhibits bacterial glucuronidase. In this connection,nialamide, isocarboxazid, and amoxapine were identified as potentinhibitors of bacterial GUS activity in purified enzyme and wholebacteria cell-based assays, but do not inhibit mammalian GUS. Thesedrugs and their average IC₅₀ values for inhibiting GUS include themonoamine oxidase inhibitors nialamide (71 nM) and isocarboxazid (128nM), the tricyclic antidepressant amoxapine (388 nM) and theantimalarial drug mefloquine (1.2 μM). These drugs had no significantactivity (75 μM to >100 μM IC₅₀) against purified mammalian GUS.Nialamide, isocarboxazid and amoxapine also showed potent activity forinhibiting endogenous GUS activity in whole E. coli cells with averageIC₅₀ values of 17, 336 and 119 nM, respectively. These drugs havepotential to be repurposed as therapeutic treatments to reduce diarrheaassociated with irinotecan chemotherapy.

The compounds of the invention are useful in eliminating or reducing thediarrhea associated with CPT-11 use for the treatment of cancer.

In yet another embodiment, the method involves screening compounds fortheir usefulness in reducing diarrhea associated with irinotecanchemotherapy. In one aspect, the method comprises: (a) assaying thecompounds for activity in inhibiting purified bacterial β-glucuronidase:(b) assaying the compounds for activity in inhibiting purified mammalianβ-glucuronidase; and (c) selecting from the compounds assayed in steps(a) and (b) a compound that inhibits the bacterial β-glucuronidase instep (a) with a potency that is more than 250-fold greater than thatwith which the compound inhibits the mammalian β-glucuronidase in step(b).

In a preferred embodiment, the assaying in step (a) comprises assayingthe compounds for activity in inhibiting purified E. colibacterial-glucuronidase. In another preferred embodiment, the assayingin step (a) comprises also assaying the compounds for activity ininhibiting endogenous β-glucuronidase activity in a culture comprisingintact bacterial cells. In yet another preferred embodiment, the intactbacterial cells are E. coli cells. In still another preferredembodiment, the assaying in step (b) comprises assaying the compoundsfor inhibiting mammalian β-glucuronidase from B. taurus.

The method for screening can further comprise administering the selectedcompound to a patient to whom irinotecan chemotherapy is being or willbe administered. In a preferred embodiment of the screening method, theselected compound generates an average IC₅₀ value of 10 μM or less in anE. coli β-glucuronidase enzyme assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Flow scheme for discovery of drugs with GUS inhibitory activity.

FIG. 2 Structures of studied drugs.

FIG. 3 Potency determinations using the E. coli GUS enzyme assay.Concentration response data for compounds was normalized to controlswith and without enzyme and plotted as percent activity. The drugsevaluated were nialamide (●), isocarboxazid (▴), phenelzine (□),amoxapine (▪), loxapine (◯) and mefloquine (♦). The average Hill slopevalues derived from the IC₅₀ curves of all active drugs ranged from0.9-1.2. Data points represent the average of three determinations perconcentration and error bars represent standard deviation. Data arerepresentative of three independent experiments.

FIG. 4 Activities in the E. coli cell-based GUS activity assay.Concentration response data for compounds was normalized to controlswith and without whole E. coli cells and plotted as percent activity.The drugs evaluated were nialamide (●), isocarboxazid (▴), phenelzine(□), amoxapine (▪), loxapine (◯) and mefloquine (♦). The average Hillslope values derived from the IC₅₀ curves of all active drugs rangedfrom 0.8-1.0. Data points represent the average of three determinationsper concentration and error bars represent standard deviation. Data arerepresentative of three independent experiments.

FIG. 5 Bacterial cytotoxicity assessment of studied drugs. E. colibacteria were treated with compounds at 10 and 100 μM, as indicated, fortwo hours. Viability was assessed with MTS viability reagent. Absorbancedata was normalized to controls with and without whole E. coli cells andplotted as percent viability, with ‘no cells’ considered as 0% viabilityand DMSO only treated cells set at 100% viable. Kanamycin, a knowncytotoxic antibiotic drug, was used as a control.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions

The following abbreviations are used in this specification:

Br=bromine

Cl=chlorine

CPT=camptothecin

DCM=Dichloromethane

DMEM=Dulbecco's Minimal Essential Media

DMF=Dimethylformamide

DMSO=Dimethylsulfoxide

DNA=deoxyribonucleic acid

F=fluorine

FPLC=fast performance liquid chromatography

H=hydrogen

HEPES=(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

I=iodine

kDal=kilodalton

MHz=megahertz

mmol=millimole

μMol=micromolar

NMR=nuclear magnetic resonance

nm=nanometer

OD=optical density

PMB=p-methoxybenzyl

PMSF=phenylmethylsulfonyl fluoride

ppm=parts per million

SDS-PAGE=sodium dodecyl sulfate polyacrylamide gel electrophoresis

TBAI=tetrabutylammonium iodide

TFA=Trifluoroacetic acid

The term “pharmaceutically acceptable salts” refers to the non-toxic,inorganic and organic acid addition salts and base addition salts ofcompounds of the present invention.

Such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,and nitric acid; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, palmoic, maleic, hydroxy-maleic, phenylacetic,glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, andisethionic acid. Pharmaceutically acceptable salts from amino acids mayalso be used. Such as salts of arginine and lysine.

Pharmaceutically acceptable salts may be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts may be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two.

As used herein, the terms “treatment” and “therapy” and the like referto alleviate, slow the progression, prophylaxis, or attenuation ofexisting disease.

As used herein, the terms “inhibit,” “inhibiting,” and the like meansthat the activity of glucuronidase is reduced.

As used herein, the term “subject” means an animal or human.

The pharmaceutical compositions of this invention comprise one or morecompounds that inhibit glucuronidase and one or more pharmaceuticallyacceptable carriers, diluents, and excipients.

Pharmaceutical compositions of the present invention may be in a formsuitable for use in this invention for examples compositions may beformulated for i) oral use, for example, aqueous or oily suspensions,dispersible powders or granules, elixirs, emulsions, hard or softcapsules, lozenges, syrups, tablets or trouches: ii) parenteraladministration, for example, sterile aqueous or oily solution forintravenous, subcutaneous, intraperitoneal, or intramuscular, iii)delivered intracerebrally or iv) topical administration, for example, asuppository or ointment.

As used herein the term “pharmaceutically acceptable” is meant that thecarrier, diluent, excipients, and/or salt must be compatible with theother ingredients of the formulation including the active ingredient(s),and not deleterious to the recipient thereof.

“Pharmaceutically acceptable” also means that the compositions, ordosage forms are within the scope of sound medical judgment, suitablefor use for an animal or human without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

A compound can also be used in the manufacture of a medicament. Thismedicament can be used for the purposes described herein.

The compositions or medicaments normally contain about 1 to 99%, forexample, about 5 to 70%, or from about 5 to about 30% by weight of thecompound or its pharmaceutically acceptable salt. The amount of thecompound or its pharmaceutically acceptable salt in the composition willdepend on the type of dosage form and the pharmaceutically acceptableexcipients used to prepare it.

The dose of the compounds of this invention, which is to beadministered, can cover a wide range. The dose to be administered dailyis to be selected to suit the desired effect. Actual dosage levels ofthe active ingredients in the pharmaceutical compositions of thisinvention may be varied so as to obtain an amount of the activeingredient, which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration without causing undue side effects or being toxic to thepatient.

The selected dosage level will depend upon a variety of factors,including the activity of the particular compound of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compounds employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

As used herein, “effective amount” and the like means the amount of thecompound or composition necessary to achieve a therapeutic effect.

An effective amount of the therapeutic compound necessary to achieve atherapeutic effect may vary according to factors such as the age, sex,and weight of the subject. Dosage regimens can be adjusted to providethe optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

Compounds of the invention can be formulated into compositions that canbe administered to a subject in need of a glucuronidase inhibitor.

The compounds or compositions thereof are used for inhibition ofglucuronidase.

The compounds or compositions thereof are used in methods for treating asubject in need of a glucuronidase inhibitor. The compounds orcompositions are administered in an amount that is effective to inhibitthe glucuronidase. In some embodiments of the invention it is ∃glucuronidase or bacterial ∃ glucoronidase that is inhibited.

The compounds or compositions described herein can be administered priorto, concurrently with or after administration of a camptothecin-derivedanticancer agent such as CPT-11. Administration of the compounds orcompositions may result in certain benefits such as decreasing the doseof the anticancer drug, increasing the tolerance of the anticancer drugand alleviating side effects from the use of the anticancer dug. Sideeffects include gastrointestinal side effects.

For purposes of the following detailed description, it is to beunderstood that the invention may assume various alternative variationsand step sequences, except where expressly specified to the contrary.Moreover, other than in any operating examples, or where otherwiseindicated, all numbers expressing, for example, quantities ofingredients used in the specification and claims are to be understood asbeing modified in all instances by the term “about”. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties to be obtained by the presentinvention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard variation found in theirrespective testing measurements.

Also, it should be understood that any numerical range recited herein isintended to include all sub-ranges subsumed therein. For example, arange of “1 to 10” is intended to include all sub-ranges between (andincluding) the recited minimum value of 1 and the recited maximum valueof 10, that is, having a minimum value equal to or greater than 1 and amaximum value of equal to or less than 10.

In this application, the use of the singular includes the plural andplural encompasses singular, unless specifically stated otherwise. Inaddition, in this application, the use of “or” means “and/or” unlessspecifically stated otherwise, even though “and/or” may be explicitlyused in certain instances.

The references cited herein are hereby incorporated by reference asfully as if set forth herein.

PREFERRED EMBODIMENTS

In a first aspect of the present invention, seventy-six (76)phenoxythiophene sulfonamides from a 35,000 compound diversity setlibrary were tested for their ability to inhibit the bacterial enzymeβ-glucuronidase. The structures and inhibitory activity of the compoundsare shown in Table 1.

Eighteen (18) analogs of BRITE-355252 were synthesized and tested toinitially explore the structural relationship these compounds displaytowards inhibition of β-glucuronidase. The structures and inhibitoryactivity of the 18 analogs of BRITE-355252 are shown in Table 2.

Compounds of formula (I)

wherein R₁, R₂, R₃, R₄ and R₅ are as defined above can be prepared by aprocess comprising the steps of

(a) reacting a halo thiophene-sulfonyl halo and R₁—N—H₂, to form aresultant N-monoprotected thiophene sulfonamide having a firstN-protecting group comprising R₁.

(b) reacting the resultant N-monoprotected amide with R₂—N-halo and acatalyst in a base, to form a resultant N,N-diprotected thiophenesulfonamide having also a second N-protecting group comprising R₂.

(c) reacting the resultant N,N-diprotected thiophene sulfonamide withCs₂CO₃ and phenol group substituted by R:

wherein R is selected from piperazin, 4-(C₁-C₄ alkyl) piperazin,4-methylpiperazin, 4-ethyl-piperazin, and 4-propylpiperazin, in asolvent, and then removing the solvent, to obtain a resultantN,N-diprotected phenoxy thiophene sulfonamide, and

(d) reacting the resultant N,N-diprotected phenoxy thiophene sulfonamidewith a deprotecting agent that is selective for deprotecting the secondN-protecting group, thereby removing the second N-protecting group, andforming a N-monoprotected phenoxy thiophene sulfonamide.

The halogen atom of the halo thiophene-sulfonyl halo compound isselected from bromine, chlorine, fluorine and iodine.

Any base that will in combination with the N-monoprotected amide withR₂—N-halo and a catalyst result in a N,N-diprotected thiophenesulfonamide can be used.

Non-limiting examples of bases that can be used are Et₃N, Na₂CO₃, K₂CO₃and NaH and any base described in the examples.

In an embodiment of the invention the halo thiophene-sulfonyl halo isdichlorothiophene-sulfonyl chloride and R1-N—H₂ is naphthylmethylamine.These groups are mixed and cooled to form a N-monoprotected thiophenesulfonamide, having a first N-protecting group that comprisesnaphthylmethyl.

In an embodiment of the invention the resultant N-monoprotectedthiophene sulfonamide, is mixed with methoxybenzyl bromide and acatalyst that can be used in a Finkelstein reaction in sodium hydride,and cooled thereby forming a N,N-diprotected thiophene sulfonamidehaving also a second N-protecting group that comprises methoxybenzyl,and the resultant N,N-monoprotected thiophene sulfonamide, and Cs₂CO₃and tert-butyl(hydroxyphenyl)piperazine-carboxylate in a solvent, aremixed and heated. The solvent is then removed to obtain a resultantN,N-diprotected phenoxy thiophene sulfonamide.

In an embodiment of the invention the resultant N,N-diprotected phenoxythiophene sulfonamide is mixed with a deprotecting agent that isselective for deprotecting the second N-protecting group, therebyremoving the methoxy benzyl that is the second N-protecting group, andthereby forming a N-alkyl or N-aryl phenoxy thiophene sulfonamide.

Examples of non-limiting embodiments of the invention are where: thedichlorothiophene-sulfonyl chloride is 4,5-dichlorothiophene-2-sulfonylchloride; the naththylmethylamine is 1-naphthylmethylamine: themethoxybenzyl bromide is 4-methoxybenzyl bromide; the catalyst istetrabutyl-ammonium iodide; the butyl(hydroxyphenyl)piperazine-carboxylate istert-butyl-4-(3-hydroxyphenyl)piperazine-1-carboxylate; the solvent isdimethyl formamide and/or the selective deprotecting agent comprisesdichloromethane and triflouroacetic acid: or a combination thereof.

In addition to dimethyl formamide, non-limiting examples of solventsthat can be used are DMSO and dioxane and the solvents described in theexamples.

The following reaction Scheme 1 illustrates the preparation of compoundswithin the scope of the present invention:

Scheme 1 refers to the preparation of compounds of formula I. Referringto Scheme 1, compounds of the formula I are prepared by reactingcommercially available 4,5-dichlorothiophene-2-sulfonyl chloride 1 withan amine to generate dichlorothiophene sulfonamide 2. PMB(p-methoxybenzyl) protected 4,5-dichlorothiophene sulfonamide 3 isgenerated by reacting compound 2 with NaH in DMF, pmethoxybenzyl bromideand a catalytic amount of TBAI. Nucleophilic displacement of the C-5chlorine with a phenol in the presence of Cs₂CO₃ produce N,N-diprotected5-(3-phenoxy)-thiophene-2-sulfonamide 3. In the final step, theprotecting group is removed using TFA in DCM (1:1) to give the desiredcompound.

In another aspect of the present invention, known compounds were assayedfor their ability to inhibit bacterial glucuronidase. The Prestwickcollection of FDA-approved drugs were screened with the GUS enzyme assayto validate the GUS enzyme assay for HTS. This screen of 1,120 compoundsresulted in 40 actives having ≥50% inhibition for a hit rate of 3.6% andall plates had Z′-factors of ≥0.8 (average Z-factor was 0.90). Since thecollection was screened at 10 μM compound, a high concentration relativeto in vivo drug levels, a cut-off of 91% inhibition was applied ascriteria for selecting initial compounds for follow-up studies. Thisrequirement allowed us to focus on the more potent actives, resulting ina short list of 7 compounds. Furthermore, antibiotics and antisepticswere eliminated since the desire is to identify drugs that do notdisrupt the gut microbial flora, but instead only inhibit bacterial GUSactivity. This further triaging of actives resulted in 4 compounds. Weobserved that two of these actives belong to the monoamine oxidaseinhibitor (MAOI) class of drugs, though another MAOI while active (62%inhibition), did not quite meet the 91% inhibition criteria. So to testmore examples of this class of inhibitors, we also included thiscompound (phenelzine) in our studies. Thus, a total of five compoundswere selected for follow-up studies which included IC₅₀ confirmation andE. coli cell-based assays. A flow chart providing an overview of thisscreening process is depicted in FIG. 1. The materials and method weused to assay these compounds are described in Example 3. infra.

The five compounds that remained after triage were nialamide,isocarboxazid, phenelzine, amoxapine and mefloquine (FIG. 1). Nialamide,isocarboxazid and phenelzine are all irreversible hydrazine-class MAOIdrugs, though nialamide is no longer on the market. Amoxapine is atricyclic antidepressant and mefloquine is an antimalarial drug.Concentration response data for each of the five compounds was generatedusing the purified E. coli GUS enzyme assay from which IC₅₀ values andHill slopes were calculated (FIG. 3). The average IC₅₀ values andstandard deviations (SDs) for the MAOIs nialamide, isocarboxazid andphenelzine were 71±31, 128±56, and 2,282±1041 nM (see Table 3 for allcompound IC₅₀ data).

Amoxapine generated an average IC₅₀ value and SD of 388±98 nM in the E.coli GUS enzyme assay. Loxapine is another tricyclic antidepressant drugthat has the identical structure as amoxapine, except that loxapine hasa methyl group, instead of hydrogen, on the secondary amine of thepiperazine ring (FIG. 1). We tested loxapine as a specificity controland this compound resulted in an average IC₅₀ value of >100 μM in theGUS enzyme assay. Thus, the methyl group on the piperazine ring ofloxapine resulted in >250-fold loss in potency. The antimalarial drugmefloquine hydrochloride generated an average IC₅₀ value of 1,212±234 nMin this GUS enzyme assay.

Compound aggregation has been reported as a common non-specificinhibitor mechanism for purified enzyme assays. The Hill slopescalculated from concentration response data can be used to eliminatemany non-specific inhibitors in enzyme assays. For single site binding,the Hill slope of an IC₅₀ curve should be 1.0. IC₅₀ curves with steepslopes, i.e. significantly greater than 1.0, can be an indicator ofnon-specific mechanisms, including compound aggregation. The IC₅₀ curvesfor all the tested compounds (with measurable IC₅₀ values) had averageHill slope values that ranged from 0.93 to 1.26 in the E. coli GUSenzyme assay, which is close to the ideal value expected when measuringinhibition of a single enzyme. To assess whether the compounds wereinhibiting signal by merely quenching fluorescence of the productformed. GUS enzyme assays were done in which compound (100 μM) was addedafter the enzyme reaction was stopped and then fluorescence was measuredas usual. Adding the compounds at the end of the assay resulted in noinhibition of signal for any of the studied compounds (data not shown),indicating that the observed activity is not due to fluorescencequenching, color quenching or other assay artifact. Thus, thesecompounds produced data consistent with specific binding to a singlesite on GUS and not inhibition by non-specific mechanisms or assayartifact.

Tumor-derived mammalian GUS activity may be important for optimalanti-tumor efficacy of irinotecan. Recent evidence suggests thatmammalian GUS may convert SN-38G back to SN-38 within the tumor and thusincrease the concentration of active drug (SN-38) in the tumor.Therefore, any inhibitor of bacterial GUS used therapeutically shouldnot inhibit the mammalian GUS since this may decrease the efficacy ofirinotecan at the site of the tumor. Therefore, we tested the three mostpotent drugs from the screen—nialamide, isocarboxazid and amoxapine—inenzyme assays identical to the E. coli GUS enzyme assay, except for theuse of mammalian GUS purified from Bovine taurus liver. Nialamidegenerated an average IC₅₀ of 74.8 μM, while isocarboxazid and amoxapinehad IC₅₀ values >100 μM. Thus, nialamide was over 1,000-fold more potentagainst E. coli GUS than mammalian GUS, while the other two drugswhere >250-fold more selective for the E. coli GUS.

An E. coli cell based assay was developed in order to assess theactivity of these drugs against whole cells, instead of purified enzyme.We took advantage of the well-known specificity and sensitivity of the4MUG substrate to detect GUS activity in E. coli cells. This assaymimicked the enzyme assay in format, with the GUS enzyme replaced bylive log-phase E. coli cells and the assay incubated for a longer time(2 hr) to detect GUS activity in these un-modified cells. Fourexperimental results confirmed that this cell-based assay was measuringGUS activity and no other E. coli cell enzymes. First, The K_(m) valuefor the substrate was determined with this cell-based assay to be 151 μM(data not shown), which is similar to the 125 μM K_(m) value wepreviously reported for the purified enzyme assay. Secondly, the Hillslopes derived from concentration-response data for all 5 active dugstested in this cell-based assay were in the 0.8-1.1 range, close to theexpected value of 1.0 for inhibition of a single enzyme. Thirdly,maximal inhibition was achieved by all active compounds (FIG. 4), whichwould be expected if 100% of the observed activity was coming from asingle enzyme rather than a mixture of enzymes. Finally, the overallrank order of potencies in the cell based assay is similar to the E.coli GUS assay and absolute IC₅₀ values derived from the cell-basedassay for the test compounds are within 5-fold of the purified bacterialGUS enzyme assay (see Table 3).

The potencies of the five hits from the Prestwick collection and the onecontrol compound were determined using the E. coli cell-based assay(FIG. 4 and Table 3). In the irreversible MOM class of drugs, nialamidepotently inhibited the cell-based assay with an IC₅₀ of 17 t 2 nM, whileisocarboxazid generated an IC₅₀ of 336±120 nM. Thus, the potency ofnialamide shifted 4-fold more potent in the cell-based assay compared tothe E. coli GUS enzyme assay, while isocarboxazid was about 3-fold lesspotent in the cell-based assay. The other irreversible MOM drug,phenelzine, was also tested in the cell-based assay and resulted in anIC₅₀ value of 7.123 nM, which is 2.6-fold less potent compared to theenzyme assay. Amoxapine was also tested in this assay resulting in anIC₅₀ of 119±61 nM, which is 3.3-fold more potent than the 388 nM IC₅₀value generated using the enzyme assay. As a specificity control,loxapine was also tested, but was completely inactive (>100 μM) in thisassay, consistent with its inactivity in the enzyme assay. Mefloquinegenerated an average IC₅₀ value of 5.961±1.526 nM and thus shifted over4.9-fold less potent in the cell-based assay compared to the enzymeassay.

One possible explanation for the inhibitory activity of the drugs in thecell-based assay is E. coli cell toxicity and/or bacteriostatic activityresulting in reduced GUS activity. Therefore, the viability ofdrug-treated E. coli cells was assessed with a metabolic viability assay(MTS kit, Promega). Each compound was tested at 100 and 10 μM for 2 hrsand data normalized to solvent (DMSO) controls with and without cells(FIG. 4). Kanamycin was used as a control to demonstrate assaysensitivity to a known bactericidal antibiotic. Kanamycin treatment (50μg/ml) reduced activity in this assay by over 90%. Amoxapine,isocarboxazid, loxapine, and nialamide showed no signs of toxicity inthis assay at up to 100 μM drug. In contrast, the 100 μM concentrationof mefloquine completely inhibited viability in this assay while the 10μM concentration was at maximum control levels (100% viability). Thus,the IC₅₀ value of mefloquine in the cell-based GUS assay may be a blendof GUS inhibitory activity and bactericidal activity at higherconcentrations. Phenelzine showed some inhibitory activity (˜10-20%) atboth the 100 and 10 μM concentrations, but not enough to explain all ofits cell-based activity.

To summarize, we screened a collection of FDA-approved drugs, thePrestwick collection, using our high throughput GUS enzyme assay. Thehit rate was high, with 40 actives displaying ≥50% inhibition at thescreening concentration of 10 μM. Raising the cut-off to 91% andelimination of antiseptics/antibiotics resulted in a short list of 4actives for follow-up. We also included a compound (phenelzine) that didnot meet the activity cut-off. It was also chosen for follow-up since itwas in the same class as two on the short list and it had >50%inhibition. Thus, the actives were nialamide, isocarboxazid, phenelzine,amoxapine and mefloquine and all of these compounds confirmed by IC₅₀determinations in the E. coli GUS enzyme assay. The five hits can becategorized into three dug classes: irreversible MAOI, tricyclicantidepressant and antimalarial.

In the irreversible MAOI class, nialamide was a very potent inhibitor ofGUS activity with an IC₅₀ of 71 nM in the GUS enzyme assay.Surprisingly, this is more potent than its reported in vitro IC₅₀ valuesof 2.6-13 μM for inhibiting monoamine oxidase (in rat brainhomogenates), its original intended target. When tested for inhibitorypotency against purified mammalian GUS, nialamide had an IC₅₀ ofapproximately 75 μM. Thus, nialamide displayed a dramatic 1,000-foldselectivity for inhibiting E. coli GUS over mammalian GUS. Furthermore,nialamide had more potent activity for inhibiting endogenous GUS in theE. coli cell-based assay, generating an IC₅₀ of 17 nM. This activity wasnot due to acute toxicity of nialamide. This unusual increased potencyin a cell-based assay (also observed with amoxapine) may be due to aunique mechanism of action or due to compound concentration inside thebacterial cell. This same phenomenon was observed previously for some,but not all, GUS inhibitor compounds. The other compound in this sameclass, isocarboxazid, was also relatively potent with an IC₅₀ of 128 nM,which is more potent than its reported potency of 4.8 μM IC₅₀ for MAO inrat brain homogenates. Isocarboxazid was >780-fold more selective forinhibiting bacterial GUS compared to its activity against mammalian GUS,which was not measurable (>100 μM IC₅₀). This dug also inhibited in thecell-based assay with an IC₅₀ of 336 nM, 2.6-fold less potent comparedto the purified enzyme assay. Phenelzine is also an MAOI that isstructurally similar to nialamide and isocarboxazid in that it containsa hydrazine group and is irreversible against its original target.Phenelzine was a much weaker inhibitor of GUS at 2.2 μM IC₅₀, incontrast to its IC₅₀ for MAO that was reported to be 70-900 nM(depending on subclass of MAO-A or MAO-B, or total activity). Thus, thephenelzine results indicated that inhibition by the MAOIs was not solelydue to the presence of a hydrazine group or the irreversible nature ofthese drugs. Phenelzine showed some toxicity to E. coli at 10 and 100μM, but not enough to account for all of its GUS inhibitory activity.

The tricyclic antidepressant amoxapine potently inhibited purified GUSwith an IC₅₀ of 388 nM. In comparison, amoxapine had no measurable IC₅₀against mammalian GUS (>100 μM) thus resulting in a >250-foldselectivity for inhibiting bacterial GUS over mammalian GUS.Furthermore, amoxapine had more potent activity in the cell-based assaywith an IC₅₀ of 119 nM. Loxapine was used as a control compound for thisclass since it has an identical structure to amoxapine except thatloxapine has a methyl group on the nitrogen of the piperazine group.Despite this very small structural difference, loxapine had nomeasurable IC₅₀ value (>100 μM) for both the GUS enzyme assay and thecell-based assay. Thus, the amoxapine/loxapine pair served to illustratethe exquisite structural selectivity for inhibiting signal in theseassays and demonstrated that a free amine in the piperazine group iscritical for inhibiting GUS.

Finally, mefloquine is an antimalarial drug that was also identified inour screen. This drug had only weak activity for inhibiting purified GUS(IC₅₀=1.2 μM) and its potency worsened by 5-fold when tested in thecell-based assay (IC₅₀=6 μM). Since this compound resulted in completeinhibition in the toxicity assay at 100 μM, though none evident at 10μM, it is possible that some of the cell-based activity is due totoxicity. Nialamide, the most potent of these drugs for inhibitingbacterial GUS, has a number of issues with respect to its use as atherapeutic. First, nialamide is no longer on the market. Nialamide waswithdrawn from the market in 1963 due to interactions with food productscontaining high levels of tyramine. Ingestion of certain foods high intyramine (e.g. aged cheese) resulted in sometimes severe tyraminetoxicity in patients taking nialamide, a general problem with all thenon-selective irreversible MAOIs. Therefore, toxicity of nialamide is amajor concern, even if it were available on the market again. However,given the high potency of this drug for inhibiting GUS, may be possibleto use lower, and thus safer, doses of nialamide that would haveacceptable side effects. Special diets, especially avoiding intake oftyramine-enriched foods, help reduce food toxicity side effects ofMAOIs. It is also conceivable that nialamide could be re-formulated forlow-dose time release in the intestine. The food-induced toxicityreported for nialamide is assumed to be due to inhibition of MAO in theintestine. Thus, we believe that dosing with nialamide may result insufficient concentrations of nialamide in the GI tract to effectivelyinhibit GUS. In contrast to nialamide, isocarboxazid is still on themarket for treatment of major depression. Like all drugs in the MAOIclass, isocarboxazid has toxicity/side effect concerns and can beproblematic in combination with many other medicines due to drug-druginteractions. Phenelzine had relatively weak activity in our assays andso it is not clear if effective in vivo concentrations could beachieved. It should be recognized that any GUS inhibitor would only beneeded short term (weeks) and perhaps even intermittently. Thus, webelieve that the long term toxicity of nialamide or isocarboxazid isavoidable with strategic, short term dosing regimens to minimize longterm drug exposure with the accompanying toxicity/side effects.

Amoxapine is a marketed member of an older class of antidepressant drugswith significantly fewer toxicity concerns compared to the MAOI drugclass. It also has a safer side effect profile and far fewer drug-druginteractions than the MAOI class of drugs in general. Antidepressant usein general is common in cancer patients and thus amoxapine could alsotreat cancer-induced depression. According to a recent report, amoxapineand loxapine have been discovered to be potent non-competitiveinhibitors of β-glycoprotein, a transporter responsible for multidrugresistance displayed by some cancer cells. Thus, the use of amoxapine asa GUS inhibitor could also have the added benefit of enhancing thesensitivity of multidrug resistant cancer cells to irinotecan and/orother chemotherapeutic drugs given in combination with irinotecan.Tricyclic antidepressants typically take about three weeks to reach peakefficacy for treatment of depression. Unlike the long term, slow actingmechanism of amoxapine for depression, amoxapine as a GUS inhibitor willonly be needed short term and should act immediately to preventre-activation of SN-38G. Thus, some of the side effects encountered withchronic use of amoxapine may be minimized with strategic intermittentdosing. The combination of its potency for inhibiting GUS and its saferprofile suggests that amoxapine is the preferred drug as a therapeutictreatment of irinotecan induced diarrhea. Moreover, based on the potencyof amoxapine for inhibiting GUS, compounds that get metabolized in vivoto form amoxapine as a metabolite would similarly be potent GUSinhibitors. In particular, loxapine undergoes metabolism that includessome of the drug being de-methylated at the piperazine ring—essentiallygenerating amoxapine and amoxapine-like molecules in vivo. Thus,loxapine, though it lacked activity in our in vitro assays, wouldsimilarly be expected to have GUS inhibitory activity in vivo due to itsmetabolites.

In short, we have identified five known drugs that inhibit E. coli GUSactivity in enzyme assays: nialamide isocarboxazid, phenelzine,amoxapine and mefloquine. These compounds displayed IC₅₀ values rangingfrom 71 nM to 2.3 μM against purified E. coli GUS. Furthermore,nialamide, isocarboxazid and amoxapine had no significant activityagainst purified mammalian GUS. All five compounds also had activity inan E. coli cell-based assay with IC₅₀ values for inhibiting endogenousGUS ranging from 17 nM to 7.1 μM.

Each of these drugs, or drugs that get metabolized in vivo to form thesedrugs as metabolites, can be administered to a patient selectively toinhibit bacterial β-glucoronidase (as compared with mammalianβ-glucoronidase) in the patient whereby to reduce diarrhea associatedwith, for example, irinotecan chemotherapy. Each of these drugs can beadministered to the patient in an amount effective to inhibit thebacterial β-glucoronidase with the dosages being controlled within thefollowing limits:

Amoxapine: less than or equal to 400 mg/day (or 600 mg/day for ahospitalized patient):

Isocarboxazid: less than or equal to 60 mg/day;

Nialamide: less than or equal to 3.3 mg/kg (from FDA web site):

Loxapine: less than or equal to 250 mg/day;

Mefloquine: less than or equal to 1250 mg/day; and

Phenelzine: less than or equal to 90 mg/day.

EXAMPLES

The invention is further understood by reference to the followingExamples, which are intended to be purely exemplary of the invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent tothose described in the Examples are within the scope of the invention.Various modifications of the invention in addition to those describedherein will become apparent to those skilled in the art from theforegoing description. Such modifications fall within the scope of theappended claims.

Example 1 β-Glucuronidase Activity Assay

Expression and Purification of E. coli β-Glucuronidase. The full-lengthE. coli β-glucuronidase gene was obtained from bacterial genomic DNA andwas cloned into the pET-28a expression plasmid (Novagen) with anN-terminal 6×-Histidine tag. BL21-DE3 competent cells were transformedwith the expression plasmid and grown in the presence of kanamycin (25ug/ml) in LB medium with vigorous shaking at 37° C. until an OD₆₀₀ of0.6 was attained. The expression was induced with the addition of 0.3 mMisopropyl-1-thio-D-galactopyranoside (IPTG) and further incubated at 37°C. for 4 hours. Cells were collected by centrifugation at 4500×g for 20min at 4° C. Cell pellets were resuspended in Buffer A (20 mM PotassiumPhosphate, pH 7.4, 25 mM Imidazole, 500 mM NaCl), along with PMSF (2μL/mL from 100 mM stock) and 0.05 μL/mL of protease inhibitorscontaining 1 mg/mL of aprotinin and leupeptin. Resuspended cells weresonicated and centrifuged at 14,500×g for 30 min to clarify the lysate.The cell lysate was flowed over a pre-formed Ni-NTA His-Trap gravitycolumn and washed with Buffer A. The Ni-bound protein was eluted withBuffer B (20 mM Potassium Phosphate, pH 7.4, 500 mM Imidazole, 500 mMNaCl). Collected fractions were then tested for initial purity bySDS-PAGE. Relatively pure (˜85%) fractions were combined and loaded intothe Åktaxpress FPLC system and passed over a HiLoad™ 16/60 Superdex™ 200gel filtration column. The protein was eluted into 20 mM HEPES, pH 7.4,and 50 mM NaCl for crystallization and activity assays. Two milliliterfractions were collected based on highest ultraviolet absorbance at 280nm. Fractions were analyzed by SDS-PAGE (which indicated >95% purity),combined, and concentrated to 10 mg/mL for long-term storage at −80° C.In addition, some experiments were performed with purified E. coliβ-glucuronidase enzyme purchased from Sigma-Aldrich.

High Throughput Screening β-Glucuronidase Assay The β-glucuronidaseassay was performed by the addition of 0.5 μl of compound (or DMSO) tothe well of a black 384-well plate followed by the addition of 30 μl ofdiluted β-glucuronidase enzyme. The enzyme was diluted in assay buffer(50 mM HEPES, pH 7.4) plus 0.0166% Triton X-100 for a final enzymeconcentration of 50 μM and final detergent concentration of 0.01%. Aftera 15 minute incubation at room temperature (23° C.), 20 ul of substrate,4-Methylumbelliferyl β-D-glucuronide hydrate (4MUG) diluted into assaybuffer, was added to the reaction for a final concentration of 125 uM.β-glucuronidase hydrolyzes the non-fluorescent 4MUG resulting in afluorescent product, 4-methylumbelliferyl. After a 30 minute incubationat room temperature, the reaction was stopped by the addition of 20 ul 1M Na₂CO₃. The fluorescence (in relative fluorescence units, RFU) wasmeasured using a 355 nm excitation filter and 460 nm emission filterusing a Victor V (Perkin Elmer) plate reader. Minimum (min) controlswere performed using reactions with no enzyme. Maximum (max) controlswere performed using no compound. 1% DMSO was maintained in allreactions. Percent inhibition was calculated using RFU data by thefollowing formula: [1−(assay readout-average of min)/(Average ofMax-Average of Min)]×100. The known-glucuronidase inhibitor, D-Glucaricacid-1,4-lactone monohydrate, was used to validate the assay and serveas a positive control. IC₅₀ value was defined as the concentration ofinhibitor calculated to inhibit 50% of the assay signal based on aserial dilution of compound. Values were calculated using either a fouror three-parameter dose response (variable slope) equation in GraphPadPrism™ or ActivityBase™. For the IC₅₀ determinations, serial dilutionsof compounds were performed in 100% DMSO with a two-fold dilution schemeresulting in 10 concentrations of compound. These results are shown inTables 1 and 2.

Example 2 Preparation of Analogs of BRITE-355252 General Procedures forthe Preparation of Analogs of BRITE-355252

All solvents and reagents were obtained from commercial sources and usedwithout further purification unless otherwise stated. All reactions wereperformed in oven-dried glassware (either in RB flasks or 20 ml vialsequipped with septa) under an atmosphere of nitrogen and the progress ofreactions was monitored by thin-layer chromatography and LC-MS.Analytical thin-layer chromatography was performed on precoated 250 prnlayer thickness silica gel 60 F₂₅₄ plates (EMD Chemicals Inc.).Visualization was performed by ultraviolet light and/or by staining withphosphomolybdic acid (PMA) or p-anisaldehyde. All the silica gelchromatography purifications were carried out by using Combiflash® Rf(Teledyne Isco) and CombiFlash® Companion® (Teledyne Isco) either withEtOAc/hexane or MeOH/CH₂Cl₂ mixtures as the eluants. Melting points weremeasured on a MEL-TEMP® capillary melting point apparatus and areuncorrected. Proton nuclear magnetic resonance (¹H NMR) spectra andcarbon nuclear magnetic resonance (¹³C NMR) spectra were recorded on aVarian VNMRS-500 (500 MHz) spectrometer. Chemical shifts (δ) for protonare reported in parts per million (ppm) downfield from tetramethylsilaneand are referenced to it (TMS 0.0 ppm). Coupling constants (J) arereported in Hertz. Multiplicities are reported using the followingabbreviations: br=broad; s=singlet d=doublet; t=triplet; q=quartet:m=multiplet. Chemical shifts (δ) for carbon are reported in parts permillion (ppm) downfield from tetramethylsilane and are referenced toresidual solvent peaks: carbon (CDCl₃ 77.0 ppm). Mass spectra wererecorded on an Agilent 1200 Series LC/MS instrument equipped with aXTerra® MS (C-18, 3.5 μm) 3.0×100 mm column.

Representative Procedure for the Preparation of4,5-Dichloro-N(Aryl/Alky) Thiophene-2-Sulfonamides

To a solution of 4,5-dichlorothiophene-2-sulfonyl chloride (1.000 g,4.002 mmol) in anhydrous CH₂Cl₂ (20 mL) was added 1-naphthylmethylamine(0.630 g, 4.007 mmol) followed by Et₃N (0.84 mL, 6.027 mmol) and stirredat room temperature for 2 h. The reaction mixture was diluted with water(20 mL) and extracted with CH₂Cl₂ (100 mL), washed with brine, dried(Na₂SO₄) and concentrated under vacuo. The residue was purified byrecrystallization from CH₂Cl₂-hexane to afford the pure4,5-dichloro-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide (1.350 g,91%) as a white crystalline product.

Representative Procedure for the PMB Protection of 4,5-dichloro-N(aryl/alkyl)thiophene-2-sulfonamides

Sodium hydride (0.081 g, 3.375 mmol) was slowly added in portions to asolution of4,5-dichloro-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide (1.250 g,3.358 mmol) in anhydrous DMF (10 mL) at 0° C., and stirred for 15 min.Then, 4-methoxybenzyl bromide (PMBBr) (0.675 g, 3.357 mmol), and acatalytic amount of TBAI (0.030 g, 0.081 mmol) were added at 0° C., andallowed to stir at room temperature for 2 h. After completion of thereaction, it was quenched by slow addition of water (5 mL) and extractedwith EtOAc (100 mL), washed with water and brine, dried (Na₂SO₄),concentrated under vacuo and the residue purified by flash silica gelcolumn chromatography (Combiflash® Rf) using EtOAc-hexane (1:9) aseluant to afford4,5-dichloro-N-(4-methoxybenzyl)-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide (1.500 g, 91%) as a white solid.

Representative Procedure for the Coupling of Phenols with PMB Protected4,5-dichloro-N-(aryl/alkyl)thiophene-2-sulfonamides

A mixture of4,5-dichloro-N-(4-methoxybenzyl)-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide(0.100 g, 0.203 mmol), tert-butyl4-(3-hydroxyphenyl)piperazine-1-carboxylate (0.068 g, 0.244 mmol) andCs₂CO₃ (0.099 g, 0.304 nmol) in anhydrous DMF (2 mL) was heated at 80°C. for 2.5 h. The solvent was removed under vacuo and the residue waspurified by Combiflash® Rf (Isco) using EtOAc-hexanes (1:9) to obtain awhite solid (0.140 g, 94%).

Representative Procedure for the Deprotection PMB Group

To a solution of tert-butyl4-(3-(3-chloro-5-(N-(4-methoxybenzyl)-N-(naphthalen-1-ylmethyl)sulfamoyl)thio-phen-2-yloxy)phenyl)piperazine-1-carboxylate(0.085 g, 0.116 mmol) in anhydrous CH₂Cl₃ (2 mL) was added TFA (2 mL)and stirred at room temperature for 3 h. The solvent mixture was removedunder vacuo and the residue was re-dissolved in CH₂Cl₂ (20 mL), washedwith aqueous sat. NaHCO₃ followed by brine, dried (Na₂SO₄), andconcentrated under vacuo.

BRITE-3552524-Chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The crude product was purified by flash silica gel column chromatographyusing MeOH—CH₂Cl₂ (1:9) to afford a light orange solid (0.055 g, 92%).¹H NMR (500 MHz, DMSO-d₆): δ (ppm): 2.81 (t, 4H, J=5.0 Hz), 3.09 (t, 4H,J=5.0 Hz), 4.56 (s, 2H), 6.43 (dd 1H, J=2.0, 8.0 Hz), 6.75 (t, 1H, J=2.5Hz), 6.83 (dd, 1H, J=2.5, 8.0 Hz), 7.26 (t, 1H, J=8.0 Hz), 7.43-7.48 (m,3H), 7.54-7.58 (m, 2H), 7.87 (dd, 1H, J=1.5, 7.5 Hz), 7.93-7.96 (m, 1H),8.06-8.09 (m, 1H). APCI/ESI MS: m/z 513.9 [M+H]⁺

BRITE-4927964-Chloro-N-methyl-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 89%% yield: White solid, mp: 144-146° C.;

¹H NMR (500 MHz, DMSO-d): δ (ppm): 2.55 (s, 3H), 2.86 (t, 4H, J=4.5 Hz),3.14 (t, 4H, J=4.5 Hz), 4.63 (s, 2H), 6.62-6.66 (m, 1H), 6.84-6.88 (m,2H), 7.30 (t, 1H, J=8.0 Hz), 7.48-7.62 (m, 4), 7.91 (s, 1H), 7.94 (d,1H, J=8.0 Hz), 7.98 (d, 1H, J=9.0 Hz), 8.29 (d, 1H, J=8.0 Hz). APCI/ESIMS m/z 527.9 [M+H]⁺

BRITE-4927944-Chloro-5-(3-(4-methylpiperazin-1-yl)phenoxy)-N-(naphthalen-1-ylmethyl)thiophene-2-sulfonamide

The product was prepared in 89%% yield: White solid, mp: 156-158C; ¹HNMR (500 MHz, DMSO-d): δ (ppm): 2.21 (s, 3H), 2.43 (t, 4H, J=5.0 Hz),3.17 (t, 4H, J=5.0 Hz), 4.55 (d, 2H, J=4.5 Hz), 6.44 (dd, 1H, J=2.0, 8.0Hz), 6.78 (t, 1H, J=2.0 Hz), 6.84 (dd, 1H, J=2.0, 8.0 Hz), 7.27 (t, 1H,J=8.0 Hz), 7.43-7.48 (m, 3H), 7.53-7.58 (m, 211), 7.88 (dd, 1H, J=1.5,7.5 Hz), 7.93-7.97 (m, 1H), 8.05-8.09 (m, 1H), 8.52 (t, 1H, J=4.5 Hz,NH). APCI/ESI MS Im/z 527.9 [M+H]⁺

BRITE-4928094-Chloro-N-(3-fluorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 75% yield, White solid, mp: 86-88° C.(decomposed): 1H NMR (500 MHz, DMSO-d): δ (ppm): 3.06 (t, 4H J=5.0 Hz),3.20 (t, 4H, J=5.0 Hz), 4.23 (s, 211), 6.54 (dd, 1H, J=2.5, 8.0 Hz),6.67 (t, 1H, J=2.5 Hz), 6.77 (dd, 1H, J=2.5, 8.0 Hz), 6.95-7.02 (m, 2H),7.04 (d, 1H, J=7.0 Hz), 7.23-7.32 (m, 211), 7.33 (s, 1H).

APCI/ESI MS m/z 481.9 [M+H]⁺

BRITE-3548734-Chloro-5-(3-(piperazin-1-yl)phenoxy)-N-(3-(trifluoromethyl)benzyl)thiophene-2-sulfonamide

The product was prepared in 71% yield: White solid, mp: 58-60° C.(decomposed): ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.03 (t, 4H, J=5.0 Hz),3.17 (t, 4H, J=5.0 Hz), 4.29 (s, 2H), 6.53 (dd, 1H, J=2.5, 8.0 Hz), 6.66(t, 1H, J=2.5 Hz), 6.76 (dd, 1H, J=2.5, 8.0 Hz), 7.22-7.25 (m, 1H), 7.31(s, 1H), 7.43-7.50 (m, 3H), 7.56 (d, 1H, J=7.0 Hz).

APCI/ESI MS m/z 531.9 [M+H]⁺

BRITE-4928084-Chloro-N-(3-methylbenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 71% yield; White solid, nip: 122-124C: ¹HNMR (500 MHz, CDCl₃): δ (ppm): 2.33 (s, 3H), 3.03 (t, 4H, J=5.0 Hz),3.17 (t, 4H, J=5.0 Hz), 4.20 (s, 2H), 6.52 (dd, 1H, J=2.5, 8.0 Hz), 6.66(t, 1H, J=2.5 Hz), 6.76 (dd, 1H, J=2.5, 8.0 Hz), 7.02 (d, 1H, J=8.0 Hz),7.04 (s, 1H), 7.11 (d, 1H, J=7.5 Hz), 7.20 (d, 1H, J=8.0 Hz), 7.23 (d,1H, J=8.0 Hz), 7.32 (s, 1H). APCI ESI-MS m/z 477.9 [M+H]⁺

BRITE-4928074-Chloro-N-(3,5-dichlorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 89% yield; Yellowish syrup: ¹H NMR (500 MHz,CDCl₃): δ (ppm): 3.06 (t, 4H, J=5.0 Hz), 3.20 (t, 4H, J=5.0 Hz), 4.19(s, 2H), 6.56 (dd, 1H, J=1.5, 8.0 Hz), 6.68 (s, 1H), 6.77 (dd, 1H,J=1.5, 8.0 Hz), 7.14 (d, 2H, J=0.5 Hz), 7.24-7.29 (m, 2H), 7.31 (s, 1H).APCI/ESI MS m/z 531.8 [M+H]⁺

BRITE-3549094-Chloro-N(4-methoxyphenyl)-S-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 89% yield; White solid, mp: 118-120° C.: ¹HNMR (500 MHz, CDCl₃+CD₃OD): δ (ppm): 3.02 (t, 4H, J=5.0 Hz), 3.16 (t,4H, J=5.0 Hz), 3.79 (s, 3H), 6.48 (dd, 1H, J=1.5, 8.0 Hz), 6.60 (t, 1H,J=1.5 Hz), 6.74 (dd, 1H, J=2.0.8.5 Hz), 6.81-6.85 (m, 2H), 7.05-7.09 (m,2H), 7.18 (s, 1H), 7.22 (t, 1H, J=8.0 Hz). APCI/ESI MS m/z 479.9 [M+H]⁺

BRITE-4928064-Chloro-N-(naphthalen-1-ylmethyl)-5-(4-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 97% yield: Light orange solid, mp: 156-158°C.: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.04-3.07 (m, 4H), 3.13-3.16 (m,4H), 4.65 (s, 2H), 6.89-6.92 (m, 2H), 7.02-7.05 (m, 2H), 7.32 (s, 1H),7.37-7.42 (m, 2H), 7.52-7.55 (m, 2H), 7.83 (dd, 1H, J=2.0, 7.0 Hz),7.86-7.89 (m, 1H), 7.92-7.94 (m, 1H).

APCI/ESI MS m/z 514.0 [M+H]⁺

BRITE-4928054-Chloro-N-(2-chlorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 83% yield; Light orange solid, mp: 99-101°C.: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.02-3.04 (m, 4H), 3.15-3.18 (m,4H), 4.35 (s, 2H), 6.49 (dd, 1H, J=2.0.8.0 Hz), 6.64 (t, 1H, J=2.0 Hz),6.75 (dd, 1H, J=2.5, 8.5 Hz), 7.20 (s, 1H), 7.22 (d, 1H, J=0.5 Hz), 7.24(s, 1H), 7.30 (s, 1H), 7.32-7.35 (m, 2H).

APCI/ESI MS m/z 497.9 [M+H]⁺

BRITE-3551234-Chloro-N-(3-chlorobenzyl)-1-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 85% yield: White solid, nip: 123-125° C.: ¹HNMR (500 MHz, CDCl₃): δ (ppm): 3.01-3.04 (m, 4H), 3.15-3.18 (m, 4H),4.21 (s, 2H), 6.54 (dd, 1H, J=2.5, 8.0 Hz), 6.67 (t, 1H, J=2.5 Hz), 6.77(dd, 1H, J=2.5, 8.0 Hz), 7.13-7.16 (m, 1H), 7.21-7.30 (m, 4H), 7.32 (s,1H), APCI/ESI MS: 497.9 [M+H]⁺

BRIT,-4928024-Chloro-N-(4-chlorobenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 78% yield, Light orange solid, mp: 118-120°C.: ¹H NMR (500 MHz, CDCl₃): δ (ppm): 3.01-3.04 (m, 4H), 3.15-3.18 (m,4H), 4.20 (s, 2H), 6.53 (ddd, 1H, J=0.5, 2.0, 8.0 Hz), 6.67 (t, 1H,J=2.0 Hz), 6.77 (dd, 1H, J=2.0, 8.0 Hz), 7.18-7.21 (m, 21), 7.22 (s,1H), 7.29-7.32 (m, 2), 7.34 (s, 1H).

APCI/ESI MS m/z 497.9 [M+H]⁺

BRITE-4928034-Chloro-N-(4-methoxybenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 48% yield; White solid, mp: 106-108C: ¹H NMR(500 MHz, CDCl₃): δ (ppm): 2.97 (t, 4H, J=5.0 Hz), 3.12 (t, 4H, J=5.0Hz), 3.77 (s, 3H), 4.14 (s, 2H), 6.51 (dd, 1H, J=2.0.8.0 Hz), 6.64 (t,1H, J=2.0 Hz), 6.73 (dd, 1H, J=2.0, 8.0 Hz), 6.82 (d, 2H, J=8.5 Hz),7.14 (d, 2H, J=8.5 Hz), 7.23 (t, 1H, J=8.5 Hz), 7.28 (s, 1H). APCI/ESIMS m/z 494.0 [M+H]⁺

BRITE-3552274-Chloro-N-(3-methoxybenzyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 31% yield; White solid, nip: 60-62° C.: ¹HNMR (500 MHz, CDCl₃): δ (ppm): 2.97 (t, 4H, J=5.0 Hz), 3.12 (t, 4H,J=5.0 Hz), 3.76 (s, 311), 4.18 (s, 2H), 6.52 (dd, 1H, J=2.0, 8.0 Hz),6.64 (t, 1H, J=2.0 Hz), 6.73 (dd, 1H, J=2.0, 8.5 Hz), 6.76 (s, 1H),6.78-6.83 (m, 2H), 7.22 (ABq. 2H, J=8.5 Hz), 7.28 (s, 1H). APCI/ESI MSm/z 494.1 [M+H]⁺

BRITE-4928004-Chloro-N-(3-chlorophenyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 78% yield; White solid, mp: 154-156° C.: ¹HNMR (500 MHz, CDCl₃): δ (ppm): 3.17 (t, 4H, J=5.0 Hz), 3.28 (t, 4H,J=5.0 Hz), 6.49 (dd, 1H, J=2.0, 8.0 Hz), 6.74 (s, 1H), 6.78 (d, 1H,J=7.5 Hz), 6.83 (dd, 1H, J=2.0, 8.0 Hz), 6.86 (dd, 1H, J=1.0, 7.5 Hz),6.96 (s, 1H), 7.10 (t, 1H, J=8.0 Hz), 7.23 (s, 1H), 7.26 (t, 1H, J=8.0Hz), 8.34 (br s, 1H, NH). APCI/ESIMS m/z 484.0 [M+H]⁺

BRITE-4927994-Chloro-N-(3-methoxyphenyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 78% yield; White solid, mp: 180-182° C.: ¹HNMR (500 MHz, CDCl₃): δ (ppm): 3.03 (t, 4H, J=5.0 Hz), 3.14 (t, 4H,J=5.0 Hz), 3.78 (s, 3H), 6.47 (dd, 1H, J=2.0.8.0 Hz), 6.59 (t, H, J=2.0Hz), 6.65-6.68 (m, 1H), 6.71-6.76 (m, 31), 7.18-7.23 (m, 2H), 7.27 (s,1H). APCI/ESI MS m/z 480.0 [M+H]⁺

BRITE-4927984-Chloro-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

To a solution of tert-butyl 4-(3-(5-(N,N-bis(4-methoxybenzyl)sulfamoyl)-3-chlorothiophen-2-yloxy)phenyl)piperazine-1-carboxylate(0.430 g, 0.602 mmol) in anhydrous CH₂Cl₂ (0.5 mL) was added TFA (4.5mL) and stirred at room temperature for 4 h. The solvent mixture wasremoved under vacuo and the residue was re-dissolved in CH₂Cl₂ (30 mL),washed with aqueous sat. NaHCO₃followed by brine, dried (NaSO₄), andconcentrated under vacuo. The residue was purified by Combiflash® Rf(Isco) using MeOH—CH₂Cl₂ (1:5) to give a white solid (0.180 g, 80%). ¹HNMR (500 MHz, CD₃OD): δ (ppm): 3.02-3.05 (m, 4H), 3.19-3.22 (m, 4H),6.56 (dd, 1H, J=2.0, 8.0 Hz), 6.73 (t, 1H, J=2.0 Hz), 6.84 (dd, 1H,J=2.0, 8.5 Hz), 7.27 (t, 1H, J=8.5 Hz), 7.40 (s, 1H).

APCI/ESI MS m/z 374.0 [M+H]⁺

BRITE-492797N-(Naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide

The product was prepared in 87% yield; Light orange solid, mp: 65-67C:¹H NMR (500 MHz, CDCl₃): δ (ppm): 2.92 (t, 4H, J=5.0 Hz), 3.08 (1, 4H,J=5.0 Hz), 4.62 (s, 2H), 6.43 (d, 1H, J=4.5 Hz), 6.56-6.60 (m, 1H), 6.65(t, 1H, J=2.5 Hz), 6.71 (dd, 1H, J=2.0, 8.5 Hz), 7.23 (t, 1H, J=8.5 Hz),7.37 (d, 2H, J=4.5 Hz), 7.39 (d, 1H, J=4.0 Hz), 7.48-7.54 (m, 2H), 7.79(t, 1H, J=4.5 Hz), 7.82-7.86 (m, 1H), 7.95 (dd, 1H, J=1.0, 7.5 Hz),APCI/ESI MS m/z 480.1 [M+H]⁺

Example 3 Materials And Methods

Material & Methods

All common reagents such as HEPES, Triton X-100, carbenicillin, anddimethyl sulfoxide (DMSO) were reagent-grade quality and obtained fromThermo Fisher Scientific (Waltham Mass.) or Sigma-Aldrich (St. Louis,Mo.). 4-methylumbelliferyl glucuronide (4MUG) was obtained fromSigma-Aldrich (St. Louis, Mo.). The solid black 96-well plates(cat#3915) for the assay and 96 well clear plates (cat#9017) forcytotoxicity assay were from Corning Incorporated (Corning, N.Y.).Falcon polypropylene plates (cat#1190) used for serial dilution ofcompounds were obtained from Becton Dickinson (Franklin Lake, N.J.).Amoxapine, nialamide, isocarboxazid and other compounds for follow-upstudies were obtained from Sigma-Aldrich. The Prestwick ChemicalCollection was obtained from Prestwick Chemical Company (WashingtonD.C.). E. coli DH5α (Zymo Research, Irvine, Calif.) was used for thecell-based assay. The expression and purification of GUS enzyme from E.coli carrying an expression plasmid containing the full-length E. coliGUS gene has been previously described (26). Bovine taurus GUS enzymewas purchased from Sigma-Aldrich. In addition, some experiments wereperformed with purified E. coli β-glucuronidase enzyme purchased fromSigma-Aldrich.

GUS Enzyme Assay—Manual Version

The semi-automated GUS high throughput enzyme assay was performed aspreviously described [25] and was used to screen the Prestwick ChemicalCollection. The follow-up studies were performed manually in a similarmanner with the exception of plate type and volumes, as briefly outlinedhere. Compound stock solutions were made in 100% DMSO. Serial dilutionsof compounds for IC₅₀ determinations were initially performed in 100%DMSO in 96 well polypropylene plates, then each compound concentrationdiluted into assay buffer (50 mM HEPES, pH 7.4 and 0.017% Triton X-100),producing a constant 5% DMSO in all wells. Subsequently, 20 μl of thisaqueous diluted compound (or just 5% DMSO for controls) was added to thewells of a solid black 96-well plate followed by 40 μl of GUS enzyme (83μM GUS) diluted in assay buffer. After addition of enzyme, the reactionwas initiated by addition of 40 μl of 4MUG substrate (312.5 μM 4MUG)diluted in 50 mM HEPES. pH 7.4, 4MUG stock solutions were prepared inthe same buffer. Final concentrations in the assembled assay were 50 mMHEPES. pH 7.4, 0.01% Triton X-100, 1% DMSO. 125 μM 4MUG and 33 μM GUS.The enzyme reaction was allowed to proceed for 20 minutes at 23° C., andwas terminated by the addition of 40 μl of a 1M sodium carbonatesolution. Fluorescence at 460 nm was determined using 355 nm excitationwavelength with a 0.1 s/well read time in a BMG PheraStar (BMG LABTECH.Cary, N.C.). Fluorescence data, expressed in relative fluorescence units(RFU), were normalized to DMSO (100% activity) and “no enzyme” (0%activity) controls as maximum and minimum responses, respectively. TheBovine taurus GUS enzyme assay was performed in an identical mannerexcept Bovine taurus GUS enzyme (1 nM) was used instead of bacterialGUS. The IC₅₀ values and Hill slopes were calculated from concentrationresponse data using GraphPad Prism software (GraphPad Software Inc., LaJolla, Calif.) employing either four-parameter or a three parameter(fixed bottom) curve fit.

GUS Cell Based Assay

Cultures of E col (DH5α) carrying the empty expression vector pCMV5 weregrown over night in LB containing carbenicilin (50 μM) and then used toinitiate fresh LB/carbenicillin cultures adjusted to an initial OD of0.1. These cultures were allowed to reach an OD of 0.6 and then washedtwice with 50 mM HEPES, pH 7.4 containing carbenicillin 50 μM, andconcentrated by centrifugation to an OD of 1 for use in the assay. TheGUS cell based assay was performed in an identical manner as the enzymeassay except the Triton X-100 was left out of the assay buffer, the E.coli cells replaced the enzyme and the reaction was allowed to proceedfor 2 hrs at 37° C. The resulting data was analyzed as outlined for theenzyme assay.

Toxicity Assay

Compounds were tested for cytotoxicity in E. coli cells. The cells weregrown and prepared for assay as described above, and plated in clear96-well plates. Cells were treated with 100 μM and 10 μM concentrations(1% DMSO) of test compounds and incubated for 2 hours at 37° C.Subsequently, 25 uL of MTS viability reagent (CellTiter 96 AqueousNon-Radioactive Cell Proliferation Assay Kit, Promega Corp., Madison.WS) was added to the wells and incubation continued for 5 minutes. Theplates were then analyzed for absorbance at 490 nm on a SpectraMax Plus384 (Molecular Devices, Sunnyvale, Calif.). Controls included DMSO only(considered 100% viability), “no cells” (representing 0% viable cells),and the cytotoxic positive control compound kanamycin at 50 μg/ml.

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TABLE 1 β-Glucuronidase Inhibitory activity of phenoxythiophenesulfonamides Compound ID Structure IC₅₀ (μm) BRITE-354972

0.000 BRITE-355123

0.090 BRITE-355252

0.090 BRITE-354975

0.120 BRITE-354989

0.150 BRITE-354909

0.170 BRITE-354725

0.190 BRITE-354969

0.200 BRITE-355417

0.210 BRITE-355006

0.210 BRITE-355017

0.220 BRITE-354979

0.230 BRITE-355004

0.240 BRITE-354667

0.270 BRITE-354966

0.270 BRITE-355262

0.280 BRITE-354965

0.290 BRITE-354873

0.290 BRITE-354615

0.310 BRITE-355016

0.320 BRITE-354517

0.320 BRITE-354958

0.330 BRITE-355360

0.340 BRITE-355227

0.350 BRITE-354948

0.390 BRITE-355336

0.410 BRITE-355423

0.430 BRITE-355468

0.500 BRITE-355202

0.510 BRITE-355003

0.510 BRITE-354946

0.560 BRITE-354983

0.570 BRITE-355014

0.600 BRITE-355015

0.630 BRITE-354627

0.710 BRITE-354764

0.730 BRITE-354993

0.730 BRITE-354956

0.750 BRITE-354984

0.750 BRITE-354974

0.760 BRITE-354947

0.770 BRITE-354392

0.790 BRITE-355045

0.880 BRITE-354994

0.900 BRITE-354957

0.920 BRITE-355074

0.970 BRITE-354565

1.000 BRITE-354998

1.000 BRITE-354955

1.310 BRITE-355296

1.370 BRITE-355008

1.390 BRITE-355005

6.700 BRITE-355192

16.140 BRITE-354839

20.030 BRITE-354428

20.560 BRITE-355224

22.910 BRITE-355018

23.330 BRITE-355240

28.570 BRITE-355329

31.510 BRITE-355250

50.060 BRITE-355339

76.560 BRITE-355319

84.560 BRITE-355243

92.780 BRITE-355180

101.980 BRITE-355244

123.470 BRITE-355214

134.850 BRITE-355030

149.400 BRITE-355234

151.550 BRITE-355169

152.600 BRITE-354458

175.430 BRITE-355211

192.490 BRITE-355201

201.630 BRITE-355233

375.580 BRITE-355221

401.950 BRITE-355253

443.900 BRITE-354502

965.440

TABLE 2 Structure and β-Glucuronidase InhibitoryActivity of BRITE-355252analogs Compound ID Structure IC50 (μM) BRITE-354873

0.030 BRITE-354909

0.060 BRITE-355123

0.020 BRITE-355227

0.050 BRITE-355252

0.020 BRITE-492794

10.170 BRITE-492796

0.120 BRITE-492797

0.090 BRITE-492798

0.330 BRITE-492799

0.070 BRITE-492800

0.070 BRITE-492802

0.030 BRITE-492803

0.100 BRITE-492805

0.040 BRITE-492806

0.300 BRITE-492807

0.130 BRITE-492808

0.030 BRITE-492809

0.040

TABLE 3 Summary of GUS Inhibitory Activity of Studied Drugs E. coli GUSB. taurus GUS E. coli Currently Enzyme Assay^(a) Enzyme Assay^(a)Cell-Based Assay^(a) Drug Drug Class Marketed IC₅₀ ± SD (nM) IC₅₀ ± SD(nM) IC₅₀ ± SD (nM) Nialamide Irreversible MAOI No  71 ± 32 74,813 ±1,841 17 ± 2  Isocarboxazid Irreversible MAOI Yes 128 ± 56 >100,000  336± 120 Phenelzine Irreversible MAOI Yes  2,282 ± 1041  ND^(b) 7,123 ±1650 Amoxapine Tricyclic Antidepressant Yes 388 ± 98 >100,000 119 ± 61 Loxaspine^(c) Tricyclic Antidepressant Yes >100,000 ND >100,000Mefloquine Antimalarial Yes 1,212 ± 234  ND 5,961 ± 1526 ^(a)IC₅₀ valuedeterminations were performed at least three times, with average IC₅₀values and standard deviations (SD) shown. The range of average Hillslopes for all measurable IC₅₀ curves (where at least 50% inhibition wasobtained) was 0.84 to 1.26. ^(b)ND = not determined; ^(c)This drug wasincluded as a study control

1-18. (canceled)
 19. A compound of formula (I)

or a pharmaceutically acceptable salt thereof wherein: each of R₁ and R₂is the same or different and is selected from H, naphthalene,naphthalene-(C₁-C₄) alkyl, naphthalene-1-ylmethyl,naphthalene-1-ylethyl, naphthalene-1-ylpropyl, 3-fluorobenzyl,3-chlorobenzyl, 3-bromobenzyl, 3-iodobenzyl, 3-(trifluoromethyl)benzyl,3-(trichloromethyl)benzyl, 3-(tribromomethyl) benzyl,3-(triiodomethyl)benzyl, 3-(C₁-C₄ alkyl)-benzyl, 3-methylbenzyl,3-ethyl-benzyl, 3-propylbenzyl, 3,5-dichlorobenzyl, 3,5-difluorobenzyl,3,5,-dibromobenzyl, 3,5-diiodobenzyl, 3-chlorophenyl, 3-fluorophenyl,3-bromophenyl, 3-iodophenyl, 3-(C₁-C₄ alkyoxy) phenyl, 3-methoxyphenyl,3-ethoxyphenyl, 3-propoxyphenyl, 4-methoxyphenyl, 4-(C₁-C₄ alkyoxy)phenyl, 4-ethoxyphenyl, 4-propoxyphenyl, 2-chlorobenzyl, 3-chlorobenzyl,4-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl,2-bromobenzyl, 3-bromobenzyl, 4-bromobenzyl, 2-iodobenzyl, 3-iodobenzyl,4-iodobenzyl, 3-(C₁-C₄ alkyoxy) benzyl; 3-methoxybenzyl,4-methoxy-benzyl, 3-ethoxybenzyl, 4-ethoxybenzyl, 3-propoxybenzyl,4-(C₁-C₄ alkyoxy)phenyl and 4 propoxybenzyl; each of R₃ and R₄ is thesame or different and is selected from H, F, Cl, Br, and I, and R₅ isselected from 3-(R-1-yl)phenyl, and 4-(R-1-yl)phenyl, wherein R isselected from piperazin, and 4-(C₁-C₄ alkyl) piperazin,4-methylpiperazin, 4-ethyl-piperazin, and 4-propylpiperazin, or apharmaceutically acceptable salt of the compound.
 20. A method oftreating a subject in need of a glucuronidase inhibitor comprisingadministering to the subject a composition comprising an amount ofcompound of claim 19 that is effective as an inhibitor of glucuonidaseactivity, or a pharmaceutically acceptable salt of the compound.
 21. Themethod of claim 20, wherein the compound is selected from one or more ofphenoxy thiophene sulfonamides, pyridine sulfonyls, benzene sulfonyls,thiophene sulfonyls, thiazole sulfonyls, thiopnene carbonyls, andthiozole carbonyls.
 22. The method according to claim 20, wherein thecompound of formula

or a pharmaceutically acceptable salt thereof wherein: each of R₁ and R₂is the same or different and is selected from H, naphthalene,naphthalene-(C₁-C₄) alkyl, naphthalene-1-ylmethyl,naphthalene-1-ylethyl, naphthalene-1-ylpropyl, 3-fluorobenzyl,3-chlorobenzyl, 3-bromobenzyl, 3-iodobenzyl, 3-(trifluoromethyl)benzyl,3-(trichloromethyl)benzyl, 3-(tribromomethyl) benzyl,3-(triiodomethyl)benzyl, 3-(C₁-C₄ alkyl)-benzyl, 3-methylbenzyl,3-ethyl-benzyl, 3-propylbenzyl, 3,5-dichlorobenzyl, 3,5-difluorobenzyl,3,5,-dibromobenzyl, 3,5-diiodobenzyl, 3-chlorophenyl, 3-fluorophenyl,3-bromophenyl, 3-iodophenyl, 3-(C₁-C₄ alkyoxy) phenyl, 3-methoxyphenyl,3-ethoxyphenyl, 3-propoxyphenyl, 4-methoxyphenyl, 4-(C₁-C₄ alkyoxy)phenyl, 4-ethoxyphenyl, 4-propoxyphenyl, 2-chlorobenzyl, 3-chlorobenzyl,4-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl,2-bromobenzyl, 3-bromobenzyl, 4-bromobenzyl, 2-iodobenzyl, 3-iodobenzyl,4-iodobenzyl, 3-(C₁-C₄ alkyoxy) benzyl; 3-methoxybenzyl,4-methoxybenzyl, 3-ethoxybenzyl, 4-ethoxybenzyl, 3-propoxybenzyl,4-(C₁-C₄ alkyoxy)phenyl and 4 propoxybenzyl; each of R₃ and R₄ is thesame or different and is selected from H, F, Cl, Br, and I, and R₅ isselected from 3-(R-1-yl)phenyl and 4-(R-1-yl)phenyl, wherein R isselected from piperazin, 4-(C₁-C₄ alkyl) piperazin, 4-methylpiperazin,4-ethyl-piperazin, and 4-propylpiperazin.
 23. A method for making acompound of formula (I):

or a pharmaceutically acceptable salt thereof of claim 19, wherein: eachof R₁ and R₂ is the same or different and is selected from H,naphthalene, naphthalene-(C₁-C₄) alkyl, naphthalene-1-ylmethyl,naphthalene-1-ylethyl, naphthalene-1-ylpropyl, 3-fluorobenzyl,3-chlorobenzyl, 3-bromobenzyl, 3-iodobenzyl, 3-(trifluoromethyl)benzyl,3-(trichloromethyl)benzyl, 3-(tribromomethyl) benzyl,3-(triiodomethyl)benzyl, 3-(C₁-C₄ alkyl)-benzyl, 3-methylbenzyl,3-ethyl-benzyl, 3-propylbenzyl, 3,5-dichlorobenzyl, 3,5-difluorobenzyl,3,5,-dibromobenzyl, 3,5-diiodobenzyl, 3-chlorophenyl, 3-fluorophenyl,3-bromophenyl, 3-iodophenyl, 3-(C₁-C₄ alkyoxy) phenyl, 3-methoxyphenyl,3-ethoxyphenyl, 3-propoxyphenyl, 4-methoxyphenyl, 4-(C₁-C₄ alkyoxy)phenyl, 4-ethoxyphenyl, 4-propoxyphenyl, 2-chlorobenzyl, 3-chlorobenzyl,4-chlorobenzyl, 2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl,2-bromobenzyl, 3-bromobenzyl, 4-bromobenzyl, 2-iodobenzyl, 3-iodobenzyl,4-iodobenzyl, 3-(C₁-C₄ alkyoxy) benzyl; 3-methoxybenzyl,4-methoxybenzyl, 3-ethoxybenzyl, 4-ethoxybenzyl, 3-propoxybenzyl,4-(C₁-C₄ alkyoxy)phenyl and 4 propoxybenzyl; each of R₃ and R₄ is thesame or different and is selected from H, F, Cl, Br, and I, and R₅ isselected from 3-(R-1-yl)phenyl and 4-(R-1-yl)phenyl, wherein R isselected from piperazin, 4-(C₁-C₄ alkyl) piperazin, 4-methylpiperazin,4-ethyl-piperazin, and 4-propylpiperazin, comprising the steps of: (a)reacting a halo thiophene-sulfonyl halo and R₁—N—H₂, wherein R₁ asdefined above to form an N-monoprotected thiophene sulfonamide having afirst N-protecting group comprising R₁, (b) reacting the resultantN-monoprotected thiophene sulfonamide with R₂—N-halo wherein R₂ is asdefined above and a catalyst in a base, forming a resultantN,N-diprotected thiophene sulfonamide having a second N-protecting groupcomprising R₂, (c) reacting the N,N-diprotected thiophene sulfonamide ofstep (b) with Cs₂CO₃; and a phenol substituted by R, wherein R isselected from piperazin, 4-(C₁-C₄ alkyl) piperazin, 4-methylpiperazin,4-ethylpiperazin, and 4-propylpiperazin, in a solvent, and then removingthe solvent, to obtain N,N-diprotected phenoxy thiophene sulfonamide,and (d) reacting the N,N-diprotected phenoxy thiophene sulfonamide witha deprotecting agent that is selective for deprotecting the secondN-protecting group, removing the second N-protecting group, and forminga N-monoprotected phenoxy thiophene sulfonamide.
 24. The method of claim23 for making a compound of formula (I) wherein: (a) the halothiophenesulfonyl halo is dichlorothiophene-sulfonyl chloride and the groupR₁—N—H is naphthylmethylamine, and the dichlorothiophene-sulfonylchloride and naphthylmethylamine, are mixed and cooled, thereby forminga N-monoprotected thiophene sulfonamide, having a first N-protectinggroup that comprises naphthylmethyl, (b) adding with mixing and coolingto the resultant N-monoprotected thiophene sulfonamide, methoxybenzylbromide and a catalyst in a base that is sodium hydride, thereby forminga N,N-diprotected thiophene sulfonamide having also a secondN-protecting group that comprises methoxybenzyl, (c) adding with mixingand heating to the resultant N,N-diprotected thiophene sulfonamide, andCs₂CO₃ and butyl (hydroxyphenyl) piperazine-carboxylate in a solvent,and then removing the solvent, to obtain a resultant N,N-diprotectedphenoxy thiophene sulfonamide, and (d) mixing the resultantN,N-diprotected phenoxy thiophene sulfonamide with a deprotecting agentthat is selective for deprotecting the second N-protecting group,thereby removing the methoxy benzyl that is the second N-protectinggroup, and forming a N-monoprotected phenoxy thiophene sulfonamide. 25.The method of claim 24, wherein (i) the dichlorothiophene-sulfonylchloride is 4,5-dichlorothiophene-2-sulfonyl chloride, (ii) thenaththylmethylamine is 1-naphthylmethylamine, (iii) the methoxybenzylbromide is 4-methoxybenzyl bromide, (iv) the catalyst istetrobutylamonium iodide, (v) the butyl (hydroxyphenyl)piperzine-carboxylate istert-butyl-4-(3-hydroxyphenyl)piperazine-1-carboxylate, (vi) the solventis dimethyl formamide, or (vii) the selective deprotecting agentcomprises dichloromethane and triflouroacetic acid; or a combination oftwo or more thereof.
 26. A method for screening compounds for theirusefulness in reducing diarrhea associated with irinotecan chemotherapy,the method comprising: (a) assaying the compounds for activity ininhibiting purified bacterial β-glucoronidase; (b) assaying thecompounds for activity in inhibiting purified mammalian β-glucoronidase;and (c) selecting from the compounds assayed in steps (a) and (b) acompound that inhibits the bacterial β-glucoronidase; in step (a) with apotency that is more than 2.50-fold greater than the compound inhibitsthe mammalian β-glucoronidase in step (b).
 27. The method according toclaim 26, wherein the assaying in step (a) comprises assaying thecompounds for activity in inhibiting purified E. colibacterial-glucoronidase.
 28. The method according to claim 27, whereinthe assaying in step (a) comprises also assaying the compounds foractivity in inhibiting endogenous β-glucoronidase activity in a culturecomprising intact bacterial cells.
 29. The method according to claim 28,wherein the selected compound generates an average IC₅₀ value of 388 nMor less in the E. coli β-glucoronidase enzyme assay.
 30. A method forinhibiting or reducing diarrhea in a patient being treated with a drugthat metabolizes to form a metabolite that is a substrate for abacterial β-glucoronidase enzyme, the method comprising administering tothe patient a compound in an amount effective to inhibit thebacterial-glucoronidase enzyme, wherein the compound is selected fromthe group consisting of nialamide, isocarboxazid, phenelzine, amoxapine,loxapine, and mefloquine.
 31. The method according to claim 30, whereinthe drug is irinotecan.
 32. The compound according to claim 1, wherein Ris 4-(C₁-C₄ alkyl) piperazin, selected from 4-methylpiperazin,4-ethyl-piperazin, and 4-propylpiperazin.
 33. A compound of the formula

or a pharmaceutically acceptable salt thereof.
 34. A compositioncomprising a compound according to claim 33 and one or morepharmaceutically acceptable carriers, diluents and excipients.
 35. Themethod of claim 20, wherein the compound is

or a pharmaceutically acceptable salt thereof.